Cardiac sarcoplasmic reticulum (SR) is regulated by diverse intracellular signals including Ca2+ and protein kinases. In the proposed work we will investigate the regulation of cardiac SR at the molecular and cellular level, with specific intention of determining the function of one family of kinase, protein kinase C (PKC). While results from any laboratories have shown that activation of PKC can produce changes in heart function, more focused studies by the Principal Investigators have shown that PKC activation can alter Ca2+ handling by the SR. Over the next five years we propose to examine broadly how PKC activation leads to altered SR function using diverse, yet complimentary methods, involving biochemical, electrophysiological, and fluorescence measurements. The experimental plan is organized around five specific experimental series. We will determine the effects of PKC activation on the Ca2+ transient and investigate how they come about. In these studies we will determine how PKC activation leads to an altered Ca2+ transient and how this might depend on altered SR function per se versus changes in Ca2+ current (ICa). These experiments will involve voltage- and current-clamped protocols with isolated adult rat ventricular myocytes. In a second related experimental series, Ca2+ transients will be stimulated by photolysis of caged Ca2+ transient under circumstances that are independent of the effects of ICa. The specificity of PKC-activating agents will be assessed by comparing results in control cells to those obtained in PKC-depleted cells. Biochemical studies will define functional substrates for PKC in cardiac SR. These experiments will take advantage of an established preparation of cultured neonatal myocytes to examine phosphorylation reactions in situ. The effects of PKC activation on the phosphorylation state of several important SR proteins will be assessed, including phospholamban, Ca2+ ATPase, and the CA2+ release channel (ryanodine receptor). More complete understanding of the actions of PKC in heart requires knowledge of the subtypes of this family of enzymes found in a cardiac myocytes. Accordingly, PKC isoforms expressed in cardiac tissue will be identified by using a novel reverse transcriptase polymerase chain reaction method recently developed by the Principal Investigators. The planned work will provide important new insight on the specific cellular and molecular mechanisms by which PKC alters SR function. Furthermore, these experiments should establish a foundation from which other important aspects of cardiac SR function can be examined and provide the understanding the insight that can lead to improved therapy for myocardial contractile dysfunction.